Pharmaceutical composition comprising albumin as an active ingredient

ABSTRACT

The present invention provides a pharmaceutical composition for treatment of corneal and conjunctival lesion, and dry eye comprising albumin as an active ingredient. The pharmaceutical composition is also useful for increase of eye surface epithelium mucin secretion. The present invention further provides a method for treatment of corneal and conjunctival lesion and dry eye, which comprises administering, to a subject in need of such treatment, albumin in an amount effective. In addition, the present invention provides a use of albumin for manufacture of a pharmaceutical composition of the present invention.

This is a division of application Ser. No. 08/981,229 filed Dec. 19,1997, which is a continuation of PCT/JP97/01329, filed Apr. 17, 1997,which continued from U.S. application Ser. No. 08/752,928, filed Nov.21, 1996, now abandoned, and U.S. application Ser. No. 08/752,941, filedNov. 21, 1996, now abandoned and which PCT application embodies thesubject matter of said U.S. applications.

Priority is claimed from Apr. 19, 1996, and Apr. 26, 1996, based onJAPAN application Nos: 098090/1996, and 106866/1996, respectively, andfrom U.S. application Ser. Nos. 08/752,928 and 08/752,941, both filedNov. 21, 1996. The priority document(s) were filed in parent applicationSer. Nos. 08/752,941 and 08/752,928.

TECHNICAL FIELD

The present invention provides a pharmaceutical composition comprisingalbumin as an active ingredient. The composition of the presentinvention is useful for treatment of the conditions such as corneal andconjunctival lesion and dry eye. The composition of the presentinvention has an ability to increase eye surface epithelium mucinsecretion and also has an ability to diffuse oil. The present inventionfurther provides methods for treatment of corneal and conjunctivallesion, treatment of dry eye conditions, and increase of eye surfaceepithelium mucin secretion using the composition.

BACKGROUND ART

Corneal and conjunctival lesion is caused by forming defects from thesurface to epithelium. The cause may include pathogenic factors such askeratoconjunctivitis sicca (dry eye), various keratoconjunctivitis,allergy and infection of microorganisms (e.g. virus, bacteria, fungus,etc.), chemical factors such as cytotoxicity by chemicals and corrosiondue to acid and alkaline, physical factors such as xerophthalmia, injurydue to foreign matter (e.g. contact lens, etc.) and hot water, and thelike. It has recently been reported that antiseptics contained in anophthalmic composition (e.g. benzalkonium chloride, chlorobutanol, etc.)and ophthalmic agents (e.g. aminoglycoside antibiotics, non-steroidalanti-inflammatory drugs, IDU, pimaricin, etc.) cause a lesion of cornealepithelium (ectocornea).

For the present, in order to treat the corneal and conjunctival lesion,chondroitin sulfate, glutathione, hyaluronic acid, fibronectin, EGF, andthe like are administered or an artificial tear solution is alsoadministered for the purpose of replenishing a tear solution, but theeffect of these treatments is not yet sufficient.

Dry eye is defined as a condition with decrease or change in quality oftear, irrespective of the presence or absence corneal and conjunctivallesion (Yamada et al. GANKI 43, 1289-1293(1992)). There are variousfactors for causing the dry eye, but no suitable method to recover thedecreased amount of tear normal has been found yet.

For the present, in order to treat the dry eye, an artificial tearsolution for the purpose of replenishing tear, and chondroitin sulfate,glutathione, hyaluronic acid, fibronectin, EGF, and the like for thepurpose of relieving subjective symptoms are administered, but theeffects are not yet sufficient.

From the recent investigation, it is believed that normal eye surfaceepithelium expresses mucin-like glycoproein and that said glycoproteintakes an active part in maintaining tear film. Furthermore, mucinsecreted from conjunctival germ cells has been known to be responsibleto stabilize tear film. So, defect of eye surface epithelium caused byany factor may induce abnormal eye mucin secretion and thereby unstabletear film. The unstable tear film may lower the interaction levelbetween the epithelium and the tear film, and thereby the lesion ofcorneal and conjunctival epithelium may become worse (Norihiko Yokoi in"GANKA. CHIRYO NO KOTSU TO OTOSHIANA (Techniques for treatment inOphthalmologic field), edited by FUMIO KOGURE, published by KABUSHIKIKAISHA NAKAYAMA SHOTEN, Tokyo, Japan pages 26-27 (1995)). From thesepoints of view, it is suggested that conditions such as corneal andconjunctival lesion or dry eye can be treated if the tear film isstabilized by increasing mucin secretion or any other mean.

Albumin is a protein that exists widely in an animal/vegetable tissue ora body fluid, such as human serum, tear solution. For example, the humanserum albumin is used for treating hypoalbuminemia, hemorrhagic shock,and the like. In the ophthalmic field, it is also known to use as astabilizer for protein preparations such as fibronectin or interferon.It has been proposed to use the preparations such as fibronectin andinterferon for treating corneal lesion (Japanese Patent Kokai No. Sho61-103838 and No. Hei 6-271478), but there is no knowledge that albuminitself is effective for treating corneal and conjunctival lesion or dryeye.

DISCLOSURE OF INVENTION

The present invention provides a pharmaceutical composition forophthalmology comprising albumin as an active ingredient. The presentcomposition is useful for treating corneal and conjunctival lesion. Thepresent composition also useful for treating dry eye.

The origin of albumin used for the composition of the present inventionis not specifically limited. When albumin has an antigenicity, however,a problem such as allergy arises and, therefore, it is not preferred.Human origin albumin, e.g. human serum albumin is preferably used.

Human serum albumin, which is purified to the purity suitable for usingnormally in medical applications, can preferably be used in the presentinvention without any particular problem. That is, those containing notless than 80% of albumin (in case of analyzing with the electrophoresis)are preferred. In order to inactivate virus, etc., those obtained byheat-treating are preferred. Particularly, human serum albumin, which iscommercially available as a drug, is preferably used.

Albumin produced by microorganism obtained by gene recombination is alsopreferably used in the present invention. The manufacturing methodaccording to the gene recombination technique is well known to personsskilled in the art. Briefly explaining, a vector containing a genecoding a desired albumin (e.g. human albumin) may be introduced into ahost cell to transform it. The transformed cell producing the desiredprotein may be selected and cultured, then, human albumin may beisolated and purified from the cultured supernatant or cell extract.Examples of the host cell include yeast, Escherichia coli, and the like,which are used ordinary by persons skilled in the art so as to produce aprotein. In view of avoiding a risk of inclusion of virus, etc., albuminas a product of such gene recombination is more preferred.

As used herein, the term "corneal and conjunctival lesion" includescorneal and conjunctival lesion caused by pathogenic factors such askeratoconjunctivitis sicca (dry eye), various keratoconjunctivitis,allergy and infection of microorganisms (e.g. virus, bacteria, fungus,etc.), chemical factors such as cytotoxicity by chemicals and corrosiondue to acid and alkaline, physical factors such as xerophthalmia, injurydue to foreign matter (e.g. contact lens, etc.) and hot water, and thelike, antiseptics contained in an ophthalmic composition (e.g.benzalkonium chloride, chlorobutanol, etc.) and ophthalmic agents (e.g.aminoglycoside antibiotics, non-steroidal anti-inflammatory drugs, IDU,pimaricin, etc.); defects of ectocornea; corneal erosion; corneal ulcer,and the like.

As used herein, the term "dry eye" includes not only simple dry eye(tear decrement) defined as "a condition with decrease or change inquality of tear, irrespective of the presence or absence of corneal andconjunctival lesion" but also various dry eye condition such asalacrima, xerophthalmia, Sjogren syndrome, dry keratoconjunctivitis,Stevens Johnson syndrome and ocular pemphigoid, blepharitis. Further,the term "dry eye" includes dry eye after cataract operation and thataccompanied with allergic conjunctivitis, as well as dry eye likecondition such as a tear decrement of VDT (Visual Display Terminal)worker and a tear decrement without any systemic symptom caused by, forexample, dry room due to air conditioning.

As used herein, the term "treatment" or "treating" refers to any meansof control of the conditions, including prevention, cure and relief ofthe conditions and arrestation or relief of development of thecondition.

The inventor further found that albumin increases eye surface epitheliummucin secretion. Therefore, the present invention further provides apharmaceutical composition for increasing eye surface mucin secretioncomprising albumin as an active ingredient.

The inventor further found that albumin has a surfactant activity todiffuses oils. It is suggested that such surfactant activity of albumincontribute to eye surface tear film stabilization.

The pharmaceutical composition of the present invention may be in adosage forms such as tablets, pills, powders, suspensions, capsules,suppositories, injection preparations, ointments, eye drops, and thelike. It is particularly preferred to locally administer eye drops.

In case of the composition of the present invention is formulated as eyedrops, the composition may contain albumin in an amount of about 1 to1000 mg/ml, more preferably about 10 to 1000 mg/ml, further preferablyabout 50 to 1000 mg/ml. The composition may further contain apharmaceutically acceptable diluent.

As used herein, the "pharmaceutically acceptable diluent" may be anydiluent which is used for ophthalmic composition known to personsskilled in the art, for example, water, physiological saline, artificialtear solution, and the like.

The pharmaceutical composition of the present invention may furthercomprise various components that used in a normal ophthalmiccomposition, such as stabilizers, sterilizers, buffering agents,isotonic agents, chelating agents, pH adjusters, surfactants, and thelike.

Examples of the stabilizer include normal L-type amino acids such asglycine and alanine, and the like, monosaccharides such as glucose andmannose, and the like, disaccharides such as sucrose and maltose, andthe like, sugar alcohols such as mannitol and xylitol, and the like, andpolysaccharides such as dextran, and the like.

Examples of the sterilizer include benzalkonium salt, chlorhexidine saltand ester of paraoxybenzoate, and the like.

Examples of the buffering agent include boric acid, phosphoric acid,acetic acid, and citric acid or a salt thereof.

Examples of the isotonic agent include sodium chloride, potassiumchloride and saccharides, and the like.

Examples of the chelating agent include sodium edetate and citric acid,and the like.

Since it is an ophthalmic composition, pH is preferably adjusted from 5to 8.

The composition may be administered in a dosage of about 1 to 100μl/eye, preferably about 10 to 50 μl/eye, and more preferably about30-50 μl/eye.

In an another aspect, the present invention also provides a use ofalbumin for manufacture of a pharmaceutical composition of the presentinvention.

In still further aspect, the present invention provides a method fortreatment of corneal and conjunctival lesion, which comprisesadministering, to a subject in need of such treatment, albumin in anamount effective.

As used herein, the term "a subject in need of such treatment of cornealand conjunctival lesion" includes both of a patient who is actuallysuffered from corneal and conjunctival lesion and a patient suspected tobe suffered from such lesion. It includes not only the patient whosecorneal and conjunctival lesion has been actually recognized but alsothe patient who is suspected of corneal and conjunctival lesion and thepatient in the state where a high possibility of occurring the conditionsuch as a patient after keratoplasty.

In still further aspect, the present invention provides a method fortreatment of dry eye, which comprises administering to a subject in needof such treatment, albumin in an amount effective. As used herein , theterm "a subject in need of such treatment of dry eye" includes both of apatient who has the dry eye condition and a patient who suspected to besuffered from dry eye.

In these methods of the present invention, albumin may be the same asthat employed in the above-described composition.

In these methods of the present invention, albumin may be administratedas the pharmaceutical composition of the present invention. Theadministration route is not limited but topical administration to eye ismost preferable.

In these methods of the present invention, "the effective amount" ofalbumin, which is an amount for the desirable treatment, may be selectedan optimum according to the patient's symptoms, age, sex, body weight,diet, other drugs used in combination and various factors which arerecognized by persons skilled in the medical field. This effectiveamount may also vary depending on kind or activity of albumin, inaddition to the above factors. Determination of the effective amount isan operation, which is usually conducted by persons skilled in the artof the medical field.

In these methods of the present invention, the pharmaceuticalcomposition may be administered in a dosage of about 1 to 100 μl/eye,preferably about 10 to 50 μl/eye and more preferably about 30 to 50μl/eye, about 1 to 20 times per day and more preferably, about 5 to 10times per day, it is not intended to limit the scope of the invention.

In these methods of the present invention, the artificial tear solution,which has hitherto been used for treating corneal and conjunctivallesion or dry eye, may be administered together with albumin. In such acase, the artificial tear solution may be administered in the amount andschedule as usual.

In still further aspect of the present invention, the present inventionprovides a method for increase of eye surface epitherium mucinsecretion, which comprises administering, to a subject in need of suchadministration, albumin in an amount effective. According to the presentmethod, mucin secretion of the eye surface epithelium may be increased,and thereby, eye surface tear film may be stabilized.

As used herein, the "a subject in need of such administration" includesboth of a patient with abnormal eye surface mucin secretion and apatient suspected to have abnormal eye surface mucin secretion due todeficient of eye surface epithelium, such as a patient of dry eye orcorneal and conjunctival lesion.

In this method of the present invention, albumin may be administrated asthe pharmaceutical composition of the present invention. Theadministration route is not limited but topical administration to eye ismost preferable.

In this method of the present invention, "the effective amount" ofalbumin, which is an amount for increase eye surface mucin secretion,may be selected an optimum amount according to the patient's symptoms,age, sex, body weight, diet, other drugs used in combination and variousfactors which are recognized by persons skilled in the medical field.This effective amount may also vary depending on kind or activity ofalbumin, in addition to the above factors. Determination of theeffective amount is an operation, which is usually conducted by personsskilled in the art of the medical field.

In this method of the present invention, the pharmaceutical compositionmay be administered in a dosage of about 1 to 100 μl/eye, preferablyabout 10 to 50 μl/eye and more preferably about 30 to 50 μl/eye, about 1to 20 times per day and more preferably, about 5 to 10 times per day, itis not intended to limit the scope of the invention.

In this method of the present invention, the artificial tear solution,which has hitherto been used for treating corneal and conjunctivallesion or dry eye, may be administered together with albumin. In such acase, the artificial tear solution may be administered in the amount andschedule as usual.

FORMULATION EXAMPLE

In the examples of this application, donated blood albumin preparationsmanufactured by The Green Cross Corporation (Osaka, Japan) were used.These albumin preparations were obtained by using an albumin fractionwhich was isolated/purified plasma of a blood donar as a raw materialaccording to a Cohn, cold ethanol fractionation method, preparingaccording to the following Formulation Example 1 and 2, andheat-treating at 60° C. for 10 hours.

    ______________________________________                                        Formulation Example 1                                                         (Albumin content: 25%)                                                        Human serum albumin   250    mg/ml                                            Acetyltriptophan sodium                                                                             5.37   mg/ml                                            Sodium caprylate      3.32   mg/ml                                            Formulation Example 2                                                         (Albumin content: 5%)                                                         Human serum albumin   50     mg/ml                                            Acetyltriptophan sodium                                                                             1.07   mg/ml                                            Sodium caprylate      0.66   mg/ml                                            ______________________________________                                    

TEST EXAMPLE 1

Three dry eye patients with corneal and conjunctival lesion (female aged64, female aged 61 and female aged 34) were administrated to their eyeswith 25% human serum albumin (albumin content: 250 mg/ml) of FormulationExample 1, 10 times per day with a dosage of 30 to 50 μl/eye. Togetherwith administration of albumin, an artificial tear solution was alsoadministered to the eyes.

An intravital stain examination was conducted before and after theadministation to estimate the degree of corneal and conjunctival lesion.As the intravital stain examination, rose bengal staining andfluorescein staining were conducted. The rose bengal staining (RB)provides an index of corneal and conjunctival lesion, and scoring wasconducted by estimating the degree of staining of nasal and aural sidesof bulbar conjunctiva and cornea, by a score of 0-3 (total scores of 9).The fluorescein staining (F) provides an index of corneal lesion, andscoring was conducted by estimating the degree of corneal lesion by ascore of 0-3. In both cases, scoring was conducted according to a vanBijsterreld's evaluation method. In both cases, the higher the numericalvalue is, the more severe the lesion is. The results are shown in Table1.

                                      TABLE 1                                     __________________________________________________________________________    Effect of albumin administration                                                        Female aged                                                                            Female aged                                                          sixty-four                                                                             sixty-one                                                                              Female aged                                       Patient   14 Days  14 days  thirty-four*                                      Administration period                                                                   Right eye                                                                          Left eye                                                                          Right eye                                                                          Left eye                                                                          7 days                                            __________________________________________________________________________    RB                                                                            Before administration                                                                   7    7   8    8   The superior limbic part is                                                   strongly stained.                                 After administration                                                                    3    3   5    5   The superior limbic part is                                                   scarcely stained.                                 Before administration                                                                   2    2   3    2   The superior limbic part is                                                   strongly stained.                                 After administration                                                                    1    1   1    1   The superior limbic part is                                                   scarcely stained                                  __________________________________________________________________________     *She was suffered from superior limbic keratoconjunctivitis in addition t     the dry eye.                                                             

TEST EXAMPLE 2

Male aged 62 with defects of ectocornea after keratoplasty

After penetrating keratoplasty of the right eye, defects of ectocorneawere continued and 0.1% Hyaleinmini (trademark) (containing 0.1% ofhyaluronic acid) was administered. However, no effect was observed and,therefore, administration of 0.1% Hyaleinmini was terminated.Thereafter, 25% human serum albumin (albumin content: 250 mg/ml) ofFormulation Example 1 was administered to the eyes 10 times per day witha dosage of 30 to 50 μl/eye for one week. One week after the beginningof administration, ectocornea is formed and defects of ectocornea wereimproved.

TEST EXAMPLE 3

Female aged 64 with defects of ectocornea after keratoplasty

After penetrating keratoplasty of the left eye, limbus transplantationand amnion transplantation, defects of ectocornea were detected by thefluorescein staining. To the eyes of this patient, 5% human serumalbumin (albumin content: 50 mg/ml) of Formulation Example 2 wasadministered 10 times per day with a dosage of 30 to 50 μl/eye for fourweeks. Two weeks after the beginning of the administration, fluoresceinstaining showed improvement in defects of ectocornea. Four weeks afterthe beginning of the administration, no staining was observed and anapparent improvement in defects of ectocornea was recognized.

TEST EXAMPLE 4

Female aged 72 with Sjogren syndrome

To the eyes of the patient with defects of ectocorneal accompanied withSjogren syndrome, Intal (trademark) (containing sodium cromoglicate),0.1% Flumetholon (trademark) (containing fluorometholone) and anartificial tear solution were administered. However, no improvement indefects of ectocornea was observed and the administration wasterminated. Then, to the eyes of the patient, 5% human serum albumin(albumin content: 50 mg/ml) of Formulation Example 2 was administered 6times per day with a dosage of 30 to 50 μl/eye for four weeks. Fourweeks after the beginning of the administration, defects of ectocorneawere not observed.

TEST EXAMPLE 5

Female aged 45 with Sjogren syndrome

To the eyes of the patient with corneal and conjunctival lesionaccompanied with Sjogren syndrome, 5% human serum albumin (albumincontent: 50 mg/ml) of Formulation Example 2 was administered 10 timesper day with a dosage of 30 to 50 μl/eye for four weeks.

Before and after the administration, an intravital stain examination(rose bengal staining and fluorescein staining) was conducted toestimate the degree of corneal and conjunctival lesion. The intravitalstain examination was conducted by applying 2 μl of a mix solutioncontaining 1% rose bengal and 1% fluorescein to the lower eyelids of thepatient using a micro-pipette accurately, making the patient to blinkseveral times, and then observing the eyes. Rose bengal staining (RB)was measured with white light of slit lamp and fluorescein staining (F)was measured with cobalt blue light. The extent of staining was scoredfrom 0 to 9. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Effects of albumin administration                                                                     After administration                                             before administration                                                                      (four weeks)                                          ______________________________________                                        RB score                                                                             right eye 7              2                                                    left eye  7              2                                             F score                                                                              right eye 9              2                                                    left eye  9              2                                             ______________________________________                                    

TEST EXAMPLE 6

Three dry eye patients (female aged 64, female aged 6 L and female aged34) were administrated with 25% human serum albumin (albumin content:250 mg/ml) of Formulation Example 1 to their eyes 10 times per day witha dosage of 30 to 50 μl/eye. Together with administration of albumin, anartificial tear solution was also administrated to the eye.

Before and after the administration, subjective symptoms of the patientswere estimated and scored. The subjective symptom includes eye ache, eyedry feeling and eye foreign body feeling. Estimation of the symptoms wasmade by the patients themselves. The worst subjective symptom was scoredas 100 point and best or normal as 0. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Improvement of subjective symptoms by administration of album                 Patient  Female, aged 64                                                                            Female, aged 61                                                                             Female,                                   term of  14 days      14 days       aged 34*                                  administration                                                                         right eye                                                                              left eye                                                                              right eye                                                                            left eye                                                                             7 days                                ______________________________________                                        before   100      100     100    100    100                                   administration                                                                after    0        30      50     50     20                                    administration                                                                ______________________________________                                         *she was suffered from superior limbic keratoconjunctivitis in addition t     the dry eye.                                                             

TEST EXAMPLE 7

To the eyes of a dry eye patient accompanying with Sjogren syndrome(female aged 74), 5% human serum albumin (albumin content: 50 mg/ml) ofFormulation Example 2 was administered 6 times per day with a dosage of30 to 50 μl/eye for eight weeks.

After eight weeks, subjective symptoms including eye ache, eye dryfeeling and eye foreign body feeling were almost perfectly removed.Administration of albumin was stopped.

TEST EXAMPLE 8

The ability of albumin to increase mucin secretion of eye epitherium wasinvestigated.

CCL cells (conjunctival epitherium cell strain) were cultured in TCM199medium (GIBCO) containing 10% (w/v) human serum albumin (SIGMA) for 24hours according to conventional cell culture condition. As a culturecontrol, the cells were cultured with the TCM 199 medium without humanserum albumin.

The cultured cells were harvested from the culture vessel usingtrypsin-EDTA, fixed with paraformaldehyde for 30 minutes and washed withphosphate buffered saline (PBS) three times. The obtained cells wereblocked (4° C., 30 min.) with normal goat: serum and the blocked cellswere reacted (4° C., 30 min.) with mouse anti-mucin antibody (Muc 1),and then washed with PBS three times. The cells further reacted (4° C.,30 min.) with FITC-labeled anti-mouse IgG antibody and then washed threetimes with PBS.

The obtained cells were measured with Epics (Colter Co.) byflow-cytometry method to determine the proportion (%) of mucingenerating cells (positive cells) to the whole cells. The result isshown in Table 4.

                  TABLE 4                                                         ______________________________________                                        increase of mucin secretion by albumin                                                      mucin generating cells(positive)                                ______________________________________                                        control (without albumin)                                                                     15.2%                                                         cultured with 10% albumin                                                                     35.5%                                                         ______________________________________                                    

According to the result, addition of albumin to the culture mediumapparently increased mucin secretion ability of conjunctival epitherium.

TEST EXAMPLE 9

Ability to diffuse oil by albumin:

10 vol % of caster oil was added to an artificial tear solution. The oiland the solution separated out and oil drops were formed. In thissystem, 10% (v/v of artificial tear solution) of 5% albumin solution wasadded, the oil diffused over the surface of the water phase and the oildrops disappeared.

According to this example, it is suggested that albumin can act as asurfactant to protect vaporization of tear solution from eye surface.

INDUSTRIAL APPLICABILITY

The pharmaceutical composition, method and use of the present inventionare useful for treatment of corneal and conjunctival lesion and dry eye.The pharmaceutical composition, method and use of the present inventionare also useful for increasing mucin secretion of eye surfaceepitherium.

What is claimed is:
 1. A method for treatment of corneal andconjunctival lesion which comprises administering, to a subject in needof such treatment, albumin in an amount effective.
 2. The methodaccording to claim 1, wherein the said albumin is human origin albumin.3. The method according to claim 1, wherein the said albumin isadministered in the form of eye drops.
 4. The method according to claim1, which comprises administering a pharmaceutical composition containing1 to 1000 mg/ml of albumin and pharmaceutically acceptable carrier.